Cellular Stress Response:
Systems-based Approach to
Toxicant Identification and Characterization: relevance
to genotoxicity testing
Ram Ramabhadran
McKim Conference, May 19, 2010
Duluth MN
Office of Research and Development
NHEERL, Integrated System Toxicology Division
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Outline
• Problem statement- current limitations
• Need for novel approaches
– 3 Rs and Tox Testing in 21st Century report
• Cellular stress response as an early indicator
of biological response
• Stress response biology and current approach
to predict adverse outcomes
• Specific application and problems in predicting
genotoxic responses to compounds
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Regulatory Challenges
• Large number of environmental compounds with limited
toxicity information
– HPVs, etc.
– 90,000 chemicals on the EPA TSCA inventory and ~9,000
chemicals used in quantities >10,000 lbs.
– 1,468 chemicals have been tested in a rodent cancer bioassay
(CPD, 2005).
– Inerts, Mixtures
• Extrapolation from model systems to human exposure
effects
• Imperative to reduce the number of animal used in
testing– 3R’s – reduce, refine and replace
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Challenge and Approach
• Need to develop cost-effective high-throughput
screening approaches to facilitate prioritization of
data-poor chemicals
• Need to reduce and refine current level of animals
required for regulatory testing
•
•
•
•
•
Need to collect data on human cell and tissues
Incorporation of ‘toxicity pathways’
Exploitation of ‘screen-able’ pathway nodes
Utility beyond prioritization?
Data needs for QSAR
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the 21st Century: A Vision and a Strategy
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Toxicity Pathways & Adaptive Stress Responses:
Canaries in the Intracellular Coalmine
Exposure
Tissue Dose
Adapted from: Toxicity Testing in the Twenty-first
Century: A Vision and a Strategy,
National Research Council. 2007.
Biologic Interaction
Perturbation
Normal
Biologic
Inputs
Biologic
Function
Adaptive Stress
Response
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Early
Cellular
Changes
Cell death, Regeneration
Cancer?
Cell
Injury
6
Morbidity and
Mortalilty
6
Prototypic Toxicity Pathways
NAS Report pp 63-64
•Years of work
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Cellular Stress Responses:
From Pathways to Prediction
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Major Stress Response Pathways
(Relatively well understood)
Oxidative Stress
Genotoxic Stress
Heat Shock
ER Stress
Hypoxia
Inflammation
Metal Response
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Adaptive Stress-Response Pathways
• Protective signaling pathways activated in response to
environmental insults such as chemical toxicity
• Present in all metazoan cells and highly conserved
• Broad indicators of early cellular toxicity (perturbation)
• Triggered at low doses before more apical effects such
as cell death or apoptosis
• Manageable number of key cellular stress pathways
identified
• Pathways mechanistically well-characterized
• Share common architecture
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10
Stress Pathway Architecture
Perturbation
Sensor
StRE
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TF
Transducers
Target Genes
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11
Integrated Response System
Transducers
MAPK
Erk
Chk2
p38
IKK
PKC
Akt
PI3K
CK2
CamK2
TKs
Plk1
PKA
ATM
Msk1
Jnk
Chk1
CK1
Sensors/TFs
Keap1
MDM2
BiP
IKB
Nrf2
p53
XBP/ATF
NFkB
hsp90
VHL
HSF1
HIF1
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???
MTF1
NFAT5
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Integration of Multiple Upstream Inputs:
Pathways to Assays
T2
T1
Sensor
StRE
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T3
T4
TF
Target
Genes
Luciferase
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Reporter Assay Construction
Pathway
TF
Reporter/responder Genes
Oxidative Stress
Nrf2
AREx7, HMOX1, NQO1
Heat Shock
HSF1
HSPA6, DNAJB1, DNAJB11
ER Stress
XBP1
ERSEx5, GRP78
Hypoxia
HIF1
HREx5, VEGF
DNA damage
p53
p53REx4, CDKN1A, MDM2, GADD45A
Inflammation
NFkB
NFkBREx5, IL8, TNFA, IL2
Metal Stress
MTF1
MT2A, MT1E
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Multi-Stress Response Strategies
(Criticality of testing dose)
No genotox pathway
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15
Compound-Specific Profiling
Simmons, et al., Toxicological Sciences 111(2), 202–225 (2009)
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Recap
• Adaptive stress response pathways share a common
exploitable architecture
• The transducer ‘layer’ of the pathways is heavily cross-wired
and plays a role in ‘non-stress’ biology
• The transcription factor/sensor complex integrates multiple
signaling inputs
• Activated TF can be measured using reporter genes that
come in two basic ‘flavors’
• Low ‘basal’ activity → high dynamic range
• Because the patterns of activation vary by compound, a
battery of such assays can be used to build compoundspecific stress response profiles
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compounds
assays
Moving Beyond Prioritization: QBAR?
rotenone
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?
dose-response
chemicals
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QBAR Concept
Supersedes QSAR, includes metal ions & contaminants, etc.
α-naphth
MMS
Metam
Iodo
HQ
Maneb
tBHQ
Nabam
CdCl2
ZnCl2
Propineb
CuCl2
OPD
B-naphth
Thiram
MeHg
1C-24DNB
pBQ
BME
EtBr
EMS
AP-1 NFkB
ARE
hsp70
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MT2A
CMV GADD153Grp94 p21
50n
M
p53
50u
M
Grp78
SV40
500u
M
inactive
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p53: Master Switch for Genotoxicity
• One of the most studied proteins ($Bs)
• Mutations or loss found in 50 % of cancerstumor suppressor
• Responds by stabilization to gentoxic stresses
(both direct and indirect)
• Causes cell cycle arrest and apoptosis
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p53: Master Switch for Genotoxicity
?
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Pluquet and Hainaut (2001) Cancer Letters 174,1–15
Activators of p53
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Pluquet and Hainaut (2001) Cancer Letters 174,1–15
P53 Signaling Pathways
Anderson and Appella (2009): In: Handbook of Cell Signaling, 2nd edition.
R. A. Bradshaw and E. A. Dennis, (Eds), Oxford: Academic Press, 2009
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Cell Cycle Arrest vs. Apoptosis
Schlereth, et al. Molecular Cell 38, 356–368, May 14, 2010
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p53:Post-translational Modifications
Anderson and Appella (2009): In: Handbook of Cell Signaling, 2nd edition.
R. A. Bradshaw and E. A. Dennis, (Eds), Oxford: Academic Press, 2009
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p53 based Genotoxicity Assays
Commercial Assays
Gentronix
Reporter
Cellumen
Antibody
InVitrogen
Reporter
Knight, et al. (2009) Regulatory Toxicology and Pharmacology 55:188–199
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p53 Binding Sites in Responder Genes
Gene
Promoter
Intron
p53RE
(Cignal)
3
N/A
GADD45A
0
1(3)
CDKN1A
(p21)
2
4 (1)
MDM2
1
N/A
RRRCWWGYYY (R = A, G; W = A,T; Y = C, T)
separated by 0–14 base pairs
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p53 Reporter Constructs
p53RE
Luciferase
Cp
GADD45A
CDKN1A
(p21)
GADD45A
Luciferase
Gp
Luciferase
Pp
GADD45A
Luciferase
CDKN1A
(p21)
CDKN1A
(p21)
Luciferase
(Cignal)
GADD45A
CDKN1A
(p21)
CDKN1A
(p21)
GADD45A
Luciferase
Luciferase
GpGi
GpPi
PpPi
PpGi
Promoter sequence
Luciferase Open Reading Frame
Intronic sequence
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•
Promoters cloned 5’ to luciferase
ORF; introns cloned 3’ to ORF.
p53 Reporter Constructs (con’t)
MDM2
p53RE
Luciferase
MpCp
GADD45A
p53RE
Luciferase
GpCp
p53RE
MDM2
Luciferase
CpMp
p53RE
GADD45A
Luciferase
CpGp
GADD45A
MDM2
Luciferase
GiMp
GADD45A
GADD45A
Luciferase
GiGp
Promoter sequence
Luciferase Open Reading Frame
Intronic sequence
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•
Promoter fused 5’ to luciferase ORF;
introns fused to promoters and
cloned 5’ to ORF.
MMS
MMS
6.0
5.0
fold change
4.0
GpGi
Cp
Gp
3.0
PpPi
GpPi
PpGi
2.0
Pp
1.0
0.0
0.01
0.1
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1
10
[compound] uM
100
1000
MMS
MMS
3.5
3
2.5
fold change
MpCp_10
2
GiGp_10
GiMp_10
GpCp_10
CpGp_10
1.5
CpMp_10
1
0.5
0
0.01
0.1
1
10
[MMS] uM
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100
1000
10000
Chloroquine
Chloroquine
3.0
2.5
fold change
2.0
GpGi
Cp
Gp
1.5
PpPi
GpPi
PpGi
1.0
Pp
0.5
0.0
0.001
0.01
0.1
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1
[compound] uM
10
100
1000
5-Fluorouracil
5-Fluorouracil
6.0
5.0
fold change
4.0
GpGi
Cp
Gp
3.0
PpPi
GpPi
PpGi
2.0
Pp
1.0
0.0
0.001
0.01
0.1
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1
[compound] uM
10
100
1000
Doxorubicin
Doxorubicin
35
30
fold change
25
GpGi
20
Cp
Gp
PpPi
15
GpPi
PpGi
10
Pp
5
0
0.0001
0.001
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0.01
0.1
[compound] uM
1
10
Dox
Doxorubicin
25
20
MpCp_10
15
fold change
GiGp_10
GiMp_10
GpCp_10
CpGp_10
10
CpMp_10
5
0
0.0001
0.001
0.01
0.1
[Dox] uM
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1
10
Cell-type Dependent Response
Cp construct: Dox
Cignal Reporter-Doxorubicin
60
50
fold change
40
HepG2
30
MCF7
A172
20
10
0
0.00001
0.0001
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0.001
0.01
[Dox] uM
0.1
1
Diversity of p53 responses
Staib, et al. (2005) Cancer Res., 65: 10255-64
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Causes for Variable Responses
• Mode of action of compound- p53 modification
– Direct vs. indirect DNA damage
• Cell type
–level of p53 and other components
• Dose - growth arrest vs. apoptosis
–Need for dose response and cytotox
• Exposure duration- temporality of activation
–Need for time course
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Current Efforts
• Identify a gene that respond to multiple stimuli
(single reporter assay)
• Use a set of responder genes that improve
coverage- possibly multiplex
• Choose appropriate cell type that give the best
response
–Lentiviral vectors
• Improve signal/noise by genetic manipulations
• Incorporate metabolism
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Luciferin-PBI3814
120
100
fold activity
80
HepG2
CYP1A1
60
CYP1A2
CYP2E1
CYP3A4
40
20
0
1A1
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p53 Activation by γ-Radiation
Hamstra et al. Cancer Research, 66, 7482 (2006)
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Utilizing In vivo Stress Assays
control
0.2uM
5uM
Blechinger SR, Warren JT Jr, Kuwada JY, Krone PH.
Developmental toxicology of cadmium in living embryos of a
stable transgenic zebrafish line.
Environ Health Perspect. 2002 Oct;110(10):1041-6.
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125uM
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Acknowledgements
Steven Simmons
US EPA NHEERL
Chun-Yang Fan (Sygenta)
Jeanene Olin
Theresa Freudenrich
NIH Chemical Genomic Center
Menghang Xia, Sunita Shukla
Ruili Huang, Chris Austin
Jim Inglese
US EPA, National Center for
Computational Toxicology
David Reif, Bob Kavlock
Keith Houck, David Dix
National Toxicology Program
Ray Tice,Kristine Witt
The Hamner Institutes
Rusty Thomas
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Open Biosystems (Thermo-Fisher)
John Wakefield, Attila Seyhan (Wyeth)
Brookhaven National Laboratory
Carl Anderson
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